Lung Cancer is the leading cause of cancer death in the United States and worldwide, with >170,000 newly diagnosed cases each year in the US and nearly a million cases worldwide (Minna et al. Cancer Cell. 1 (1):49-52 (2002)). Despite aggressive approaches made in the therapy of lung cancer in the past decades, the 5-year survival rate for lung cancer remains under 15% (Minna et al. Cancer Cell. 1 (1):49-52 (2002)). Lung cancers are divided into two groups: non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). NSCLC (75-80% of all cancers) consists of three major types: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma (Minna (2002)). Lung carcinomas and squamous cell carcinomas represent 6-70% of all lung cancers. Surgery, chemotherapy, and radiation have been used with generally unsatisfactory results in advanced disease. Improvement in the efficacy of lung cancer treatment is a major public health goal.
Malignant pleural mesothelioma (MPM) is a highly aggressive and challenging cancer arising primarily from the pleural lining of the lung. Approximately 3,000 patients are diagnosed with MPM in the United States annually and the incidence of this tumor is predicted to increase dramatically over near term, peaking around 2020 (Thatcher, Lung Cancer 45 Suppl 1:S1-2 (2004)). Since MPM usually presents at an advanced stage, a curative resection is rarely possible. Radiotherapy has failed to show clinical benefit as a single treatment modality, and the administration of chemotherapy is mostly restricted to the advanced stage with limited efficiency (Kindler, Lung Cancer 45 Suppl 1:S125-7 (2004)). Alternative strategies based on pleural injections of recombinant cytokines have similarly proven unsatisfactory (Bard et al. Lung Cancer 45 Suppl 1:S129-31 (2004)). Since current interventions offer only limited benefit, and overall survival is low, there is an urgent need to develop new therapeutic agents based on a greater understanding of MPM's underlying molecular mechanisms.
Molecular pathogenesis of lung cancer and MPM includes alterations of expression and function of multiple genes, involving dominant oncogenes and recessive tumor suppressor genes, and abnormalities in cell signaling transduction pathways. A better understanding of molecular mechanisms for lung cancer and MPM pathogenesis should improve the treatment of patients with lung cancer.
The Wingless-type (Wnt) family of secreted glycoproteins is a group of signaling molecules broadly involved in developmental processes and oncogenesis (Polakis, Genes Dev. 14:1837-51 (2000); Lustig et al. J. Cancer Res. Clin. Oncol. 129:199-221 (2003)). Nineteen human Wnt proteins have thus far been identified. Transduction of Wnt signals is triggered by the binding of Wnt ligands to two distinct families of cell-surface receptors: the frizzled (Fz) receptor family and the LDL-receptor-related protein (LRP) family (Akiyama, Cytokine Growth Factor Rev. 11:273-82 (2000)). Intracellularly, Wnt signaling activates disheveled (Dvl) proteins, which inhibit glycogen synthase kinase-3β (GSK-3β) phosphorylation of β-catenin leading to its cytosolic stabilization. Stabilized β-catenin then enters the cell nucleus and associates with LEF/TCF transcription factors. β-catenin-Tcf/Lef induces transcription of important downstream target genes, many of which have been implicated in cancer. In the absence of Wnt signals, free cytosolic β-catenin is incorporated into a complex consisting of Axin, the adenomatous polyposis coli (APC) gene product, and glycogen synthase kinase (GSK)-3β. Conjunctional phosphorylation of Axin, APC, and β-catenin by GSK-3β designates β-catenin for the ubiquitin pathway and degradation by proteasomes (Uthoff et al., Int J Oncol 19 (4):803-10 (2001); Matsuzawa et al., Mol Cell 7 (5):915-26 2001)).
Disheveled (Dvl) is a positive mediator of Wnt signaling positioned downstream of the frizzled receptors and upstream of β-catenin. GSK-3 phosphorylates several proteins in the Wnt pathway and is instrumental in the downstream regulation of β-catenin. Mutations in the gene APC are an initiating event for both sporadic and hereditary colorectal tumorigenesis. APC mutants are relevant in tumorigenesis, since the aberrant protein is an integral part of the Wnt-signaling cascade. The protein product contains several functional domains acting as binding and degradation sites for β-catenin. Mutations that occur in the amino-terminal segment of β-catenin are usually involved in phosphorylation-dependent, ubiquitin-mediated degradation and, thus, stabilize β-catenin. When stabilized cytoplasmic-catenin accumulates, it translocates to the nucleus interacting with the Tcf/Lef high-mobility group of transcription factors that modulate expression of oncogenes such as c-myc.
It is known that Wnt/β-catenin signaling promotes cell survival in various cell types (Orford et al., J Cell Biol 146 (4):855-68 (1999); Cox et al., Genetics 155 (4):1725-40 (2000); Reya et al., Immunity 13 (1):15-24 (2000); Satoh et al., Nat—Genet. 24 (3):245-50 (2000); Shih et al., Cancer Res 60 (6):1671-6 (2000); Chen et al., J Cell Biol 152 (1):87-96 (2001); Ioannidis et al., Nat—Immunol 2 (8):691-7 (2001)). Wnt signaling pathway is also thought to be associated with tumor development and/or progression (Bienz et al., Cell 103 (2):311-20 (2000); Cox et al., Genetics 155 (4):1725-40 (2000); (Polakis, Genes Dev 14 (15):1837-51 (2000); You et al., J Cell Biol 157 (3): 429-40 (2002)). Aberrant activation of the Wnt signaling pathway is associated with a variety of human cancers, correlating with the overexpression or amplification of c-Myc (He et al., Science 281 (5382):1509-12 (1998); Miller et al., Oncogene 18 (55):7860-72 (1999); Bienz et al., Cell 103 (2):311-20 (2000); (Polakis, Genes Dev 14 (15):1837-51 (2000); Brown, Breast Cancer Res 3 (6):351-5 (2001)). In addition, c-Myc was identified as one of the transcriptional targets of the β-catenin/Tcf in colorectal cancer cells (He et al., Science 281 (5382):1509-12 (1998); Miller et al., Oncogene 18 (55):7860-72 (1999); You et al., J Cell Biol 157 (3): 429-40 (2002)).
In addition to the Wnt ligands, a family of secreted Frizzled-related proteins (sFRPs) has been isolated. sFRPs appear to function as soluble endogenous modulators of Wnt signaling by competing with the membrane-spanning Frizzled receptors for the binding of secreted Wnt ligands (Melkonyan et al., Proc Natl Acad Sci USA 94 (25):13636-41 (1997)). sFRPs can either antagonize Wnt function by binding the protein and blocking access to its cell surface signaling receptor, or they can enhance Wnt activity by facilitating the presentation of ligand to the Frizzled receptors (Uthoff et al., Int J Oncol 19 (4):803-10 (2001)). sFRPs seem to modulate apoptosis susceptibility, exerting an antagonistic effect on programmed cell death. To date, sFRPs have not yet been linked causatively to cancer. However, sFRPs are reported to be hypermethylated with a high frequency in colorectal cancer cell lines and this hypermethylation is associated with a lack of basal sFRP expression (Suzuki et al., Nat Genet 31 (2):141-9 (2002)).
Another protein called Dickkopf (Dkk) is also found to interfere with Wnt signaling and diminish accumulation of cytosolic β-catenin (Moon et al., Cell 88 (6):725-8 (1997); Fedi et al., J Biol Chem 274 (27):19465-72 (1999)). Dkk-1 antagonizes Wnt-induced signals by binding to a LDL-receptor-related protein 6 (LRP6) adjacent to the Frizzled receptor (Nusse, Nature 411 (6835):255-6 (2001)). Overexpression of Dkk-1 is also found to sensitize brain tumor cells to apoptosis (Shou et al., Oncogene 21 (6):878-89 (2002)).
The effects of Wnt proteins on cell proliferation and tumor growth seem to depend on Wnt proteins interacting with their cognate cell surface receptors and subsequently inducing downstream signaling. With Wnt proteins being secreted ligands antibodies may be used to interfere with or inhibit Wnt binding to its cell surface receptor and thus affect downstream signaling. Several antibodies against Wnt proteins have been generated. For example, anti-Wnt-1 (G-19) (sc-6280; Santa Cruz Biotechnology, Inc.) and anti Wnt2 (H-20) (sc-5208; Santa Cruz Biotechnology, Inc.) are goat polyclonal antibodies raised against peptides mapping near the N-terminus of human Wnt-1 and Wnt2 proteins, respectively. Wnt2 (V-16) is a goat polyclonal antibody raised against a peptide mapping within an internal region of Wnt2 of human origin (sc-5207; Santa Cruz Biotechnology, Inc.).
However, the use of polyclonal and monoclonal antibodies in humans is severely restricted when the polyclonal monoclonal antibodies are produced in a non-human animal. Repeated injections in humans of a “foreign” antibody, such as a mouse antibody, may lead to harmful hypersensitivity reactions, i.e., human anti-mouse antibody (HAMA) or an anti-idiotypic, response. The HAMA response makes repeated administrations ineffective due to an increased rate of clearance from the patient's serum and/or allergic reactions by the patient.
Attempts have been made to manufacture human-derived monoclonal antibodies using human hybridomas. Unfortunately, yields of monoclonal antibodies from human hybridoma cell lines are relatively low compared to mouse hybridomas. Additionally, human cell lines expressing immunoglobulins are relatively unstable compared to mouse cell lines, and the antibody producing capability of these human cell lines is transient. Thus, while human immunoglobulins are highly desirable, human hybridoma techniques have not yet reached the stage where human monoclonal antibodies with the required antigenic specificities can be easily obtained. Thus, antibodies of non-human origin have been genetically engineered to create chimeric or humanized antibodies. Such genetic engineering results in antibodies with a reduced risk of a HAMA response compared to that expected after injection of a human patient with a mouse antibody. For example, chimeric antibodies can be formed by grafting nor-human variable regions to human constant regions (Khazaeli et al. (1991), J. Immunotherapy 15:42-52). Generally humanized antibodies, are formed by grafting non-human Complementarity Determining Regions (CDRs) onto human Framework Regions (FRs) (See, Jones et al. (1986), Nature 321:522-525; and Reichman et al. (1988), Nature 332:323-327). Typically, humanized monoclonal antibodies are formed by grafting all six (three light chain and three heavy chain) CDRs form a non-human antibody into Framework Regions (FRs) of a human antibody (e.g., see, U.S. Pat. No. 6,407,213). Alternately, Fv antibodies (See, U.S. Pat. No. 4,642,334) or single chain Fv (SCFV) antibodies (See, U.S. Pat. No. 4,946,778) can be employed to reduce the risk of a HAMA response.
Despite recent advances in the understanding of Wnt2 signaling, the role of this pathway in oncogenesis is unclear. Thus, the prior art fails to provide clear evidence that compounds that modulate the Wnt2 pathway could be useful for example, for the treatment of cancer. The present invention addresses these and other needs.